This invention relates in general to separation and detection techniques and, in particular, to a system for electrokinetic separation and detection.
Capillary separation techniques such as capillary electrophoresis (CE) involving gel or liquid columns have become important analytical techniques for the analysis of various complex sample mixtures. The sample mixtures analyzed are typically in tiny volumes, such as a few nanoliters or picoliters. When the samples are available in such tiny volumes, it is necessary to employ detection methods with high sensitivity. A number of detection methods have been in use. These include absorbance, fluorescence, chemiluminescence, radioactivity, mass spectrometric and electrochemical detection. Electrochemical detection can be amperometric or conductivity-based. Electrochemical detection has certain advantages over the other detection schemes that have been developed for CE. These include sensitivity (down to the attomole level) and selectivity for amperometric, and universality for conductivity, as known to those skilled in the art. An additional advantage in electrochemical detection is that a variety of compounds of interest (e.g., carbohydrates, amino acids, alcohols, etc.) can be detected without prior derivatization. It also utilizes relatively inexpensive instrumentation.
In CE, the separation electric field applied can range from 100 volts per cm to 1,000 volts per cm. With such a high magnitude electric field inside the separation column, one of the main problems in electrochemical detection is the isolation of the detection apparatus such as measuring electrodes and the associated electronics from the separation electric field. If the detection apparatus is not properly isolated from the separation electric field, the amperometric and conductivity measurements will be less sensitive to sample components at low concentration. A number of solutions have been proposed for this problem. In "Capillary Zone Electrophoresis with Electrochemical Detection," by Wallingford and Ewing, Anal. Chem., 1987, 59, 1762-1766, a post-column electrochemical detection scheme is proposed for CE by grounding the separation capillary before detection. For this purpose, a porous glass coupler was built to connect two pieces of capillary together, where sample separation occurs in one piece of the capillary and detection occurs in the other piece of the capillary. This procedure is manually intensive and also degrades separation efficiency. End-column detection has also been proposed to minimize the effects of the separation voltage on CE-electrochemical detection. See Huang et al., Anal. Chem., 1991, 63, 189 and U.S. Pat. No. 5,126,023. In end-column detection, the detection or measuring electrode is placed outside or at the end of the separation column. When the detection or measuring electrodes are placed at or near the end of the separation column, the background noise can be substantial due to the high current in the separation column where such background noise may overwhelm measurements to be taken. For this reason, high resistivity buffers are frequently used in conjunction with the end-column detection scheme to reduce both the amount of current at the end of the column and the background noise. The requirement of high resistivity buffers, however, limits the type of buffers that can be used and therefore the separation capabilities of CE as applied to certain samples. In both the post-column and end-column detection schemes, detection noise has been observed to increase with an increase in separation voltage.
From the above, none of the solutions proposed for the problem of isolating the detection apparatus from the separation electric fields is entirely satisfactory. It is therefore desirable to provide an improved capillary electrophoretic system where the above-described difficulties are alleviated or eliminated.